The goal of this application is to develop a customized molecular platform for prenatal diagnosis that combines our proprietary padlock probe technology with the power of next-generation sequencing. The molecular platform combines comprehensiveness with customization more effectively than currently available methods, and promises to reduce per sample cost of goods to below $100. The technology enables simultaneous detection of Mendelian disorders, aneuploidy, uniparental disomy, and unbalanced structural abnormalities. Sequence capture is performed multiplexed in a single tube, in conjunction with any next-generation sequencing platform. Because the method is easy to customize, molecular geneticists will be able to add new alleles or create customized pools for certain at-risk ethnic populations. Once they are synthesized, individual probe freezer stocks can be mixed to create customized pools for virtually any future application. This application proposes to optimize the padlock probe technology and then build a 200,000-plex probe pool for prenatal diagnostics. First, we propose to map the genome coordinates for disease alleles from 18 Mendelian congenital disorders of interest to the prenatal specialist. Second, we propose to build a 1,000-plex pilot probe pool that will be used to optimize probe design, synthesis, and sequence capture. Third, we propose to use these optimized to synthesize a 200,000-plex probe pool specifically for prenatal diagnostics. Finally, we will validate the molecular technology on archival genomic DNA, amniotic fluid, and fetal nucleated red blood cells (FNRBCs). Eventually, we will market the technology to laboratories performing routine amniotic screening as well as noninvasive prenatal diagnostics. The probe pool could also be used generically for any routine molecular karyotyping application, such as cancer diagnostics or preimplantation genetic diagnosis. PUBLIC HEALTH RELEVANCE: We are developing new methods that will help to detect genetic problems before babies are born. Current methods are expensive and not customizable. We are using new technology for DNA sequencing that may one day make this process easier for geneticists.